Mechanical ventilation (MV) is used in millions of patients each year to support the respiratory system during surgery and in critically ill patients requiring ventilator support. Although MV is often a life-saving intervention, prolonged MV promotes problems in weaning patients from the ventilator. While several factors can contribute to difficult weaning, weak inspiratory muscles are a major factor. In this regard, MV results in diaphragmatic inactivity resulting in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (known as ventilator-induced diaphragm dysfunction (VIDD)). Although MV-induced diaphragmatic atrophy occurs due to both increased protein breakdown and decreased protein synthesis, proteolysis plays a dominant role. It is established that all four major proteolytic systems are activated in the diaphragm during prolonged MV and evidence indicates that the ubiquitin- proteasome system, calpains, and caspase-3 all contribute to VIDD. While the fourth major proteolytic system, autophagy, is activated in the diaphragm during prolonged MV, it remains unknown if autophagy promotes VIDD or serves as a protective mechanism by removing damaged proteins and organelles. HYPOTHESIS: Guided by preliminary results, we predict that prevention of MV-induced increases in autophagy in the diaphragm will protect against VIDD and will suppress the activation of key proteolytic systems (i.e., calpain and caspase-3) that contribute to VIDD. APPROACH: We will test this hypothesis using a well-established animal model of MV and innovative molecular tools to prevent MV-induced increases in autophagy in the diaphragm. Specifically, cause and effect will be determined by using an adeno-associated virus vector for gene delivery to express a dominant negative mutation of autophagy gene 5 that will prevent activation of autophagy in the diaphragm during prolonged MV. To determine if the impact of increased autophagy on VIDD is time dependent, we will expose animals to varying durations of MV. SPECIFIC AIMS: Aim 1 will test the hypothesis that prevention of MV-induced increases in autophagy in the diaphragm is protective against VIDD. Aim 2 will test the hypothesis that prevention of MV-induced autophagy in the diaphragm will prevent the activation of other major proteolytic systems (i.e., calpain and caspase-3) that contribute to VIDD. SIGNIFICANCE: VIDD is an important clinical problem because inspiratory muscle weakness is a major cause of the inability to wean patients from the ventilator. Our proposed experiments will provide new and important information regarding the role that autophagy plays in VIDD. Collectively, these experiments can lead to new therapeutic strategies in the prevention of VIDD.